In addition to hematopoietic stem cells, bone marrow contains “stromal cells” which are mesenchymal precursor cells (Friedenstein, A. J. et al., Exp. Hemat. 4:267-274 (1976) which is incorporated herein by reference) that are characterized by their adherence properties when bone marrow cells are removed and put on to plastic dishes. Within about four hours, stromal cells adhere to the plastic and can thus be isolated by removing non-adhered cells form the dishes. These bone marrow cells that tightly adhere to plastic have been studied extensively (Castro-Malaspina, H. et al., Blood 56:289-301 (1980); Piersma, A. H. et al., Exp. Hematol 13:237-243 (1985); Simmons, P. J. and Torok-Storb, B., Blood 78:55-62 (1991); Beresford, J. N. et al., J. Cell. Sci. 102:341-351 (1992); Liesveld, J. L. et al., Blood 73:1794-1800 (1989); Liesveld, J. L. et al., Exp. Hematot 19:63-70 (1990); and Bennett, J. H. et al., J. Call. Sci. 99:131-139 (1991)) which are incorporated herein by reference. As used herein, the term “adherent cells” is meant to refer to stromal cells and the term “non-adherent cells” is meant to refer to hematopoietic precursor cells.
Stromal cells are believed to participate in the creation of the microenvironment with the bone marrow in vivo. When isolated, stromal cells are initially quiescent but eventually begin dividing so that they can be cultured in vitro. Expanded numbers of stromal cells can be established and maintained. Stromal cells have been used to generate colonies of fibroblastic adipocytic and osteogenic cells when cultured under appropriate conditions. If the adherent cells are cultured in the presence of hydrocortisone or other selective conditions populations enriched for hematopoietic precursors or osteogenic cells are obtained (Carter, R. F. et al., Blood 79:356-364 (1992) and Bienzle, D. et al., Proc. Natl. Acad. Sci USA, 91:350-354 (1994)) which are incorporated herein by reference.
There are several examples of the use of stromal cells. European Patent EP 0,381,490, which is incorporated herein by reference, discloses gene therapy using stromal cells. In particular, a method of treating hemophilia is disclosed. Stromal cells have been used to produce fibrous tissue, bone or cartilage when implanted into selective tissues in vivo (Ohgushi, H. et al., Acte. Orthop. Scand. 60:334-339 (1989); Nakahara, H. et al. J. Orthop. Res. 9:465-476 (1991); Niedzwiedski, T. et al., Biomaterials 14:115-121 (1993); and Wakitani, S. et al., J. Bone & Surg. 76A:579-592 (1994)). In some reports, stromal cells were used to generate bone or cartilage in vivo when implanted subcutaneously with a porous ceramic (Ohgushi, H. et al. Acta. Orthop. Scand. 60:334-339 (1989)), intraperitoneally in a diffusion chamber (Nakahara, H. et al. J. Orthop. Res. 9:465-476 (1991)), percutaneously into a surgically induced bone defect (Niedzwiedski, T. et al. Biomaterials. 14:115-121 (1993)), or transplanted within a collagen gel to repair a surgical defect in a joint cartilage (Wakitani, S. et al. J. Bone & Surg. 76A:579-592(1994)). Piersma, A. H. et al. Brit. J. Hematol. 54:285-290 (1983) disclose that after intravenous bone marrow transplantation, the fibroblast colony-forming cells which make up the hemopoietic stroma lodge and remain in the host bone marrow. Stewart et al. (Blood 81:2566-2571 (1993)) recently observed that unusually large and repeated administrations of whole marrow cells produced long-term engraftment of hematopoietic precursors into mice that had not undergone marrow ablation. Also, Bienzle et al. (Proc. Natl. Acad. Sci USA, 91:350-354 (1994)) successfully used long-term bone marrow cultures as donor cells to permanently populate hematopoietic cells in dogs without marrow ablation. In some reports, stromal cells were used either as cells that established a microenvironment for the culture of hematopoietic precursors (Anklesaria, PNAS USA 84:7681-7685 (1987)) or as a source of an enriched population of hematopoietic stem cells (Kiefer, Blood 78(10):2577-2582 (1991)).